Name: design and use of a low cost, automated morbidostat for adaptive evolution of bacteria under antibiotic drug selection
Uploaded: 2016-09-27T15:00:00
Description: Watch this Scientific Journal Video about Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection at JoVE.com

The authors would like to thank Prof. Sze-Bi Hsu and Ms. Zhenzhen for useful discussions and help in the theoretical analysis and numerical simulation. Y. T. Y. would like to acknowledge funding support from the Ministry of Science and Technology under grant numbers MOST 103-2220-E-007-026 and MOST 104-2220-E-007-011, and from the National Tsing Hua University under grant numbers 103N2042E1, 104N2042E1, and 105N518CE1.

1.大会和预测试的Morbidostat设备的<ol><li>在Morbidostat的大会<ol><li>按机器上的文化小瓶用18G的注射器针头帽3孔。切三块聚乙烯管材在〜长度为7厘米插入这三件聚乙烯管上的盖。 </li><li>用带子把盖的边缘以作为铸造的聚二甲基硅氧烷（PDMS）混合物。通过用牙签手动搅拌混合将5g组分和0.5g在150毫升的塑料容器中的PDMS B成分的。加载混合物到10ml注射器中。 <ol><li>倒在与注射器的帽与PDMS混合物。烤整个...

<ol>
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从低成本分量的低足迹morbidostat设备是证明。由设备注册的耐药水平的增加是与以前的报告5一致。设计为耐药性的进化研究，该装置是可能适用于许多其他的实验。首先，药物诱导的突变的综合数据库可为一大组的临床相关抗生素来建立。例如，多药耐药性的进化途径可以通过简单地增加的实验中所用的药物的数量进行研究。这里所报告的模块的主要优点是它的灵活配置性，可实现大型?...

自推出第一种抗生素药物青霉素，微生物的耐药性已经发展成为一个全球性的健康问题1。尽管抗生素抗性的获得可在体内回顾性研究，这些实验的条件通常不会在整个演化2控制。另外，自适应进化实验室可以从抗生素药物3显示环境下的应力或选择压力微生物物种的分子进化。最近，抗生素耐药性的许多良好控制的进化实验已经阐明抗生素耐药性的出现。例如，奥斯汀的小组展示了在一个适当设计的微流体隔间4环境迅速出现。最近开发的morbidostat诱导药物的选择压力下5,6系统性的突变。该morbidostat，微生物SELEC该连续调整抗生素浓度维持几乎恒定的人口和灰装置，是从在微生物学7,8中使用的波动测试的一大进步。在波动测试，抗生素药物以高浓度注入，幸存的突变体进行筛选并计数。相反，在morbidostat微生物都在不断的挑战，并获得多个突变。 该morbidostat功能类似恒，由诺维克和Szliard于1950年发明了一种培养装置，通过同时稀释微生物种群连续9提供营养保持一个恒定的人口。自推出以来，恒化已提前和完善。目前的微流控恒化已经达到了纳升和单细胞的能力。然而，这些装置不适合于适应进化实验，这需要许多突变事件10,11的大细胞群体。近日，迷你用约10 mL工作体积恒化器也已开发以补升规模的生物反应器和微流体恒化器12,13之间的间隙。 在这里，我们提出了一种抗生素耐药性的研究设计和使用成本低，自动化morbidostat。所提出的模块可以在摇床培养箱，在微生物实验室通过更少的硬件要求使用。开源固件也很容易适合于适应进化的特定的应用，如代谢工程3。最后，morbidostat被集成到抗生素药敏试验14复用微流体平台。

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			<td>tapered shape dim: 10 mm x 4mm</td>
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			<td height="42" style="height:42px;width:275px;">Glass scintillation 20ml vial</td>
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			<td>Pyrex glass 28mm(dia.)x 61 mm(h)</td>
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			<td height="48" style="height:48px;width:275px;">Culture vial holder</td>
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			<td height="21" style="height:21px;width:275px;">Medium bottle</td>
			<td style="width:135px;">VWR</td>
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			<td height="41" style="height:41px;width:275px;">Inverted microscope</td>
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			<td>ValuePlastics.com</td>
			<td style="width:163px;">MTLL230-1</td>
			<td>used for microfluidic control</td>
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			<td>ValuePlastics.com</td>
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			<td height="21" style="height:21px;">Female luer-lock fittings to 10-32 threaded port</td>
			<td>ValuePlastics.com</td>
			<td style="width:163px;">KFTL-1</td>
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			<td height="21" style="height:21px;">NPN darlington transistor 500mA, 40V (2N6427)</td>
			<td>DigiKey.com</td>
			<td>2N6427GOS-ND</td>
			<td>used for microfluidic control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10kOhm, carbon film resistor, 0.25W</td>
			<td>DigiKey.com</td>
			<td>P10KBACT-ND</td>
			<td>used for microfluidic control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tantalum capacitor, 10uF, 25V, 10%</td>
			<td>DigiKey.com</td>
			<td>478-1841-ND</td>
			<td>used for microfluidic control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Andor CCD camera</td>
			<td style="width:135px;">Andor</td>
			<td style="width:163px;">Zyla 4.2 Plus SCMOS</td>
			<td>used for microfluidic on chip imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">ELISA plate reader</td>
			<td style="width:135px;"></td>
			<td style="width:163px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">two component Silicone&#160;</td>
			<td style="width:135px;">Momentive</td>
			<td style="width:163px;">RTV 615</td>
			<td>used for microfluidic chip fabrication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">SU-8 photoresist</td>
			<td style="width:135px;">Micrchem</td>
			<td style="width:163px;">SU8 2015</td>
			<td>used for microfluidic chip fabrication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">AZ4620 photoresist</td>
			<td style="width:135px;">Clariant</td>
			<td style="width:163px;">AZ 4620</td>
			<td>used for microfluidic chip fabrication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Plasma cleaner</td>
			<td style="width:135px;">Harrick Plasma</td>
			<td style="width:163px;">PDC 32G</td>
			<td>used for microfluidic chip fabrication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">20 Gauge Syringe Needle</td>
			<td style="width:135px;">BD</td>
			<td style="width:163px;"></td>
			<td>used for microfluidic chip fabrication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Labcycler</td>
			<td style="width:135px;">Sensoquest</td>
			<td style="width:163px;">Labcycler</td>
			<td>PCR&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">DNA polymerase</td>
			<td style="width:135px;">Toyobo</td>
			<td style="width:163px;">KDO Plus</td>
			<td>PCR amplification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Trimethoprim</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:163px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Plate reader</td>
			<td style="width:135px;">Biotek</td>
			<td style="width:163px;">Synergy H1 hybrid&#160;</td>
			<td>antibiotic resistane measurement</td>
		</tr>
	</tbody>
</table>

design and use of a low cost, automated morbidostat for adaptive evolution of bacteria under antibiotic drug selection

我们描述用于表征抗生素耐药性的进化途径一种低成本，可配置morbidostat。的morbidostat是连续监测细菌的生长并动态调整的药物浓度，因为它们进化获得抗药性不断挑战细菌细菌培养装置。该器件具有的〜10毫升的工作容积，并且完全自动化，并配备了光学密度测量结果和微型泵为介质和药物递送。为了验证平台，我们测得的逐步收购大肠杆菌 MG 1655耐甲氧苄啶，以及集成设备与复用微流体平台来研究细胞形态及药敏。该方法可以按比例放大的抗生素耐药性的实验室研究，并可扩展至适应性进化为代谢工程和其它细菌培养试验菌株改进。

上述morbidostat在图1中概略显示。在共同morbidostat操作，包括实验进化，药敏试验和细胞形态的检查，在大肠杆菌进行了验证大肠杆菌 MG1655文化受到甲氧苄氨嘧啶（TMP），常用的抗生素药物5,6。 TMP诱导耐药性非常鲜明逐步增加，并且该突变围绕二氢叶酸还原酶（DHFR）基因簇。因此，TMP是用于验证morbidostat动作的高效标准药物。每一天，微?...

Watch this Scientific Journal Video about Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection at JoVE.com

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

We describe a low cost, configurable morbidostat that enables the characterization of antibiotic drug resistance by dynamically adjusting the drug concentration. The device can be integrated with a multiplexed microfluidic platform. The approach can be scaled up for laboratory antibiotic drug resistance studies.

设计与低成本的使用，自动Morbidostat细菌的适应性进化在抗生素药物的选择

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial ...

Research

JoVE Journal

Genetics

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

程序的微生物实验室自适应演化的恒化

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

科研

遗传学

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

通过重叠2方法高通量识别协同药物组合

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

多拷贝产质粒在抗生素耐药性演变中的作用试验

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

全基因组对真核生物转录误差的监测

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

可抽税抗生素耐药盒 P1 转导制备大肠杆菌中的基因缺失

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol ...

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes

qkat: 杀手细胞免疫球蛋白样受体基因的定量半自动分型

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

基于细菌选择和生长的盘状细胞基因工程

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER

使用 Ever 设计自动化、高通量、连续细胞生长实验

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring ...

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

在彗星分析中使用冷冻组织评估DNA损伤

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Use of Drosophila S2 Cells for Live Imaging of Cell Division

使用果蝇S2细胞进行细胞分裂的活成像

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a ...